Both the phospholipid-coated microdroplet and the phospholipid-coated microcrystal depend on the membrane-forming and amphipathic properties of phospholipids to maintain their structure. As described in the previous Specification (Haynes, U.S. application Ser. No. 07/514,012 now U.S. Pat. No. 5,091,188), fatty acids and detergents are also amphipathic, but do not form membranes. Phospholipids are the major building block of biological membranes, and are very tissue compatible. An important and abundant example is lecithin (phosphatidylcholine). In the presence of excess water, phospholipids form membranes of bimolecular thickness. The polar head groups are oriented to the water; the fatty acyl chains form a palisade structure, with their ends abutting in the center of the membrane.
Liposomes, aqueous core vesicles formed from membrane-forming phospholipids such as lecithin, were first described by Bangham, Standish & Watkins (in J. Mol. Biol. 13:238, 1965). Liposomes produced by homogenization are multi-lamellar, with concentric bilayer membranes. Liposomes produced by sonication are small and unilamellar phospholipid vesicles as described by Haung (in Biochem. 8:344, 1969). Liposomes have the ability to entrap polar and highly-charged molecules in their aqueous interiors. Publications describing the use of liposomes to entrap and deliver water-soluble drugs appeared in the early and mid-1970's (cf. Gregoriadis: "The Carrier Potential of Liposomes in Biology and Medicine", New. England Journal of Medicine 295:704-710, 1976). A large number of patents have been granted for entrapment of water-soluble drugs and proteins (Papahadjopoulos, U.S. Pat. No. 4,078,052, 1978; Schneider, U.S. Pat. No. 4,089,801, 1978; Miller & Djordjevich, U.S. Pat. No. 4,133,874, 1979; Papahadjopoulos et al., U.S. Pat. No. 4,235,871, 1980; Weber et al., U.S. Pat. No. 4,38,052, 1984; Deamer, U.S. Pat. No. 4,515,736, 1985; Jizomoto, U.S. Pat. No. 4,762,720, 1988; Farmer & Beissinger, U.S. Pat. No. 4,776,991, 1988; Yagi et al., U.S. Pat. No. 4,756,910, 1988; Lenk et al., U.S. Pat. No. 5,030,453 are a small fraction of the available examples). However, most of these liposome inventions rely on complicated methods of preparation, including dissolution in organic solvents and evaporation, treatment with detergents and the like. Furthermore, the intra-vesicular space as described in these publications is always less than 10% of the total aqueous space. Thus the "stability of the entrapment" is a serious consideration since slow permeation of the entrapped molecules while the preparation is on the shelf will result in 90% of the molecules eventually being outside of the liposomes, with loss of the intended benefit of the encapsulation.
In the course of working with the phospholipid-coated microcrystal system, which incorporates phospholipid up to 20% (w/v), it became apparent to me that injectable, pharmaceutically-acceptable liposome preparations encapsulating over 50% of a water-soluble drug can be made by the simple methods of homogenization, sonication or high shear. At this concentration the stability of entrapment during storage is not an issue since the probability of molecules diffusing in is equal to the probability of molecules diffusing out.
In the present invention I propose the incorporation of particles of iron oxide in the phospholipid-coated microcrystal. The use of iron oxide in pharmaceutical systems has already been described. Widder and Senyei (U.S. Pat. No. 4,345,588, 1982) described the IV injection of albumin microspheres consisting of drug, serum albumin and Fe.sub.3 O.sub.4 powder in a ratio of 10:125:36. The albumin is crosslinked by formaldehyde. Particle diameter was 10 um. The phospholipid-coated microcrystal described by me previously (Haynes, U.S. application Ser. No. 07/514,012 now U.S. Pat. No. 5,091,188) does not rely on crosslinked albumin. Morris (U.S. Pat. No. 4,331,654, 1982) described a lyophilized preparation of &lt;3 um diameter magnetically-localizable microspheres consisting of a core of magnetite (Fe.sub.3 O.sub.4) coated with a solidified mixture of fatty acid and non-ionic detergent, and containing lecithin as a minor constituent. The phospholipid-coated microcrystal described by me previously (Haynes, U.S. application Ser. No. 07/514,012 now U.S. Pat. No. 5,091,188) does not rely on detergent to suspend the particles.
In this specification I also describe the use of the phospholipid-coated microcrystal and phospholipid-coated microdroplet as vaccine adjuvants. Much use has been made of phospholipids and oils in adjuvant systems, but no published system fits the description of the phospholipid-coated microcrystal or microdroplet. There is a considerable amount of published work on the use of liposomes as adjuvants (Allison & Gregoriadis, U.S. Pat. No. 4,053,585, 1977; Nerome et al., U.S. Pat. No. 4,826,687, 1989) and detergent-solublized oils as adjuvants (Gerber, U.S. Pat. No. 4,806,350, 1989). In the cases where phospholipids and oils have been used together, the systems contained detergents or contained high concentrations of ethylene glycol, propylene glycol or the like (Cantrell, U.S. Pat. No. 4,806,352, 1989; Cantrell & Rudbach, U.S. Pat. No. 4,803,070, 1989).